Towards improved methods for detection of Xylella fastidiosa in plant material using triplex TaqMan PCR and NGS analysis
Xylella fastidiosa is a gram-negative bacterial plant pathogen with a wide host range covering over 300 plant species. In Europe, the pathogen is causing severe disease problems since 2013, when the olive decline syndrome, caused by a specific strain of X. f. subsp. pauca, was found for the first time in the Southern part of Italy. More recently introductions in France and Spain were detected on different host plants, caused by various X. fastidiosa subspecies. Also interceptions of various subspecies in imported ornamental plants were diagnosed in Europe, among them Olea europaea, Coffea arabica and Nerium oleander. The host range of the pathogen can vary dependent on the subspecies and even the strain. Therefore the availability of subspecies or strain specific detection methods are of high importance in management strategies. In this study we combined two specific and sensitive TaqMan assays previously designed for X. fastidiosa with an internal control into a triplex TaqMan assay. The triplex TaqMan was compared with a single TaqMan assay often used for X. fastidiosa. Recently the usefulness of Next Generation Sequence (NGS) analysis was demonstrated in the field of diagnostics. Several DNA extracts from naturally infected or artificially inoculated hosts plants or plants from interceptions from France, Austria, The Netherlands, Italy and Spain, were evaluated for the presence of Xf using NGS and compared with the results of the triplex TaqMan PCR.